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We're considering organizing some interlaboratory work on calibrating luminescence reporters (e.g., luciferase), and one of the key questions I don't know the answer to is whether most plate readers can measure luminescence or not.

From a first principles perspective, any fluorescence plate reader ought to be able to measure luminescence as well - just don't turn on the excitation light source. Thus, I would expect that a typical fluorescence reader should also be able to measure luminescence.

On the other hand, searching online, I am swamped by manufacturers eager to sell me specialized luminescence readers that emphasize their sensitivity, which leads me to think that maybe typical fluorescence plate readers can't measure luminescence.

My question, then, is this: should I expect that a typical current fluorescence plate reader will also be able to measure luminescence, or is this a much more specialized capability?

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As the other answers here say, technically plate readers which are capable of fluorescent measurements can also make luminescent readings, but the sensitivity may be low (https://www.biotek.com/resources/technical-notes/use-of-the-fluorescence-optics-to-measure-luminescent-reactions-in-high-well-density-microplates-measuring-luminescence-in-1536-well-microplates/).

What I haven't seen mentioned is that there is potentially another hardware limitation depending on what type of luminescence you're using. Broadly there are two types: glow and flash luminescence.

Glow luminescence is probably most similar to fluorescence in that it builds gradually and can remain stable over an extended period (think of a lightbulb being turned on and warming up). Flash luminescence, however, is much more transient as the name might suggest. Reactions which cause flash luminescence produce a short burst of light (which could last seconds to minutes) which is typically brighter than that produced by glow luminescence. (https://www.bmglabtech.com/what-is-the-difference-between-flash-and-glow-luminescence-assays/)

As some flash luminescence assays will only produce a signal for a few seconds after the reagent is added, it is important to have immediate measurement, which can be provided by using auto-injectors within the plate reader. (https://www.biotek.com/products/detection-flash-luminescence-technology.html) These may or may not be included in a plate reader dedicated to fluorescent readings.

So to summarise, while luminescence can be measured by plate readers which can measure fluorescence, the sensitivity may be too low. In addition, extra hardware requirements, such as auto-injectors, may be required for certain luminescence assays.

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  • $\begingroup$ Do you know if luciferase is flash or glow? I'm guessing it's glow, since otherwise it wouldn't be so popular, but I hadn't heard of flash before your answer. $\endgroup$ – jakebeal Mar 12 at 11:00
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    $\begingroup$ It depends on the type of luciferase and the reagents/conditions used. I know from experience that bacterial luciferase (the lux operon) can produce a stable signal in vivo. There are luciferase assays using firefly luciferase for both flash and glow (thermofisher.com/order/catalog/product/16174 and thermofisher.com/order/catalog/product/16176) $\endgroup$ – Brad0440 Mar 13 at 13:57
  • $\begingroup$ Fascinating --- I didn't realize there was so much variety in luciferase. $\endgroup$ – jakebeal Mar 13 at 17:01
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I am René Inckemann and I am PhD student at the Max Planck Institute in Marburg, Germany. In my PhD I am using luminescence on a regular basis and I was also responsible to organize some of our Platereaders.

So therefore I can tell you that you need a Platereader which is able to measure luminescence and you can not simply take a fluorescence platereader. The hardware for measuring luminescence and fluorescence is different.

However many Platereader models in these days already come with both options for the hardware, which means that you can measure luminescence and fluorescence in the same device. You just have to check your platereader model if it is capable of measuring luminescence.So you don't need a seperate platereader just for luminescence, but you need one, which has the luminescence addon installed.

Regarding your idea for normalizing luminescence, I also have strong interest in that and we have already started working on that. Maybe we can exchange ideas at some point?

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    $\begingroup$ Do you know if the hardware difference is primarily about sensitivity, or something else? $\endgroup$ – jakebeal Mar 11 at 15:39
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Anecdotally, all of the fluorescence capable plate readers that I have used have also been luminescence capable (BMG and Tecan models).

If access to luminescence capable plate readers is restrictive, we have also been able to measure luminescence using a GelDoc designed for imaging electrophoresis gels. This may be something to explore since I imagine gel imagers are more widespread that plate readers.

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A priori what you say about the fluorescence and luminescence readers is correct, even wikipedia states this directly:

Luminescence detection is simpler optically than fluorescence detection because luminescence 
does not require a light source for excitation or optics for selecting discrete excitation 
wavelengths.

However, it is worth comparing the fluorescence and the luminescence devices sensitivity and saturation threshold. It might well be that a luminescence device, designed to detect very weak levels of luminescence is blinded (i.e., saturated) by stronger light. What is more relevant to your case, the detectors in a fluorescence device might be insufficiently sensitive for practical purposes of luminescence study.

Still, it is better to consult a real expert before spending money on a new piece of equipment.

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